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Non-protein coding RNAs perform essential functions in living organisms. They commonly exist as dynamic ensembles of conformational states. While many structurally known RNAs are trapped in one or a few conformations by interactions with proteins or tertiary contacts between stems, bulges, or loops the knowledge of equilibrium structures, conformational space, and tertiary conformational changes of large RNAs not being restrained by external or tertiary interactions is still very limited.Helical four-way and three-way junctions (4WJs and 3WJs) are an essential structural motif of the for functional RNA structures. Here we explore the topology of a set of a 4WJ and related 3WJs related to the hairpin ribozyme by measuring more than 250 different FRET-pairs using single-molecule multi-parameter fluorescence detection [1]. Using FRET restrained high-precision structural modeling combined with full atom MD simulations as a hybrid tool [2,3], we resolve the structures of three coexisting conformers of a fully Watson-Crick base paired RNA4WJ. By a suitable choice of the number of bases in the bulges the helices arrangements of the corresponding 3WJs can span a huge conformational space which is necessary for the stem communication in functional RNAs.[1] Sisamakis, E., et al.; Methods in Enzymology 475, 455-514 (2010).[2] Sindbert, S., et al.; J. Am. Chem. Soc. 133, 2463-2480 (2011).[3] Kalinin, S. et al. Nat. Methods 9, 1218-1225 (2012)

Forster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling is a powerful tool for analyzing biomolecular structures. Here we apply multi-parameter fluorescence detection (MFD) of single molecules and ensemble Time-Correlated Single Photon Counting measurements to perform FRET study on RNA three- and four-way-junctions (4WJs and 3WJs) with a high level of precision in distance better than 1% of the Forster radius [1].We have generated a database of RNA 4WJs and six different RNA 3WJs with different bulges and sequences to study the influence of these factors on the junction conformations of RNA 3WJs. Overall 260 FRET pairs were measured with single-molecule MFD at 20 mM MgCl2 concentration and analyzed with the analysis toolkit [2] that includes probability distribution analysis (PDA) for FRET distance determination and FRET position and screening (FPS) toolkit for structural model generation.Monte Carlo simulations showed that sterically allowed conformational space for RNA junctions is large. However, FRET measurements detect the existence of three different static conformers for RNA 4WJ, whereas RNA 3WJs have only one predominant static conformation. terically allowed conformational space for RNA is large. Their junction geometry was described in terms of mutual and Euler angles between helices. The FRET-derived structures suggest that the sequence dictates a junction specific conformation within the large topology space. Furthermore we see that bulges in the junction region determine orientation and rotation of helices and induce coaxial stacking between two of them.[1] Antonik, M., et al., J.Phys.Chem.B, 110, 6970-6978 (2006)[2] Kalinin, S. et al, Nat. Meth., 9, 1218-1225 (2012)

Like for many other non-coding RNAs, helical four-way and three-way junctions (4WJs and 3WJs) are an essential structural motif of the for functional RNA structures. Using FRET restrained high-precision structural modeling as a hybrid tool we resolve the structures of three coexisting conformers of a fully Watson-Crick base paired RNA4WJ based on the hairpin ribozyme. 51 different FRET-pairs were measured using single-molecule multi-parameter fluorescence detection (smMFD). For each dataset, the single-molecule approach allowed for the simultaneous extraction of three distances (and their corresponding errors) belonging to one major FRET state and two minor states. Distinct Mg2+-affinities were used for the assignment of the two minor states to the corresponding conformers. Rigid body models for the major and both minor conformers were obtained by docking rigid ds A-RNA helices explicitly taking into account dye position distributions. The three rigid body models were refined by all-atom MD simulations and coarse-grained RNA folding using FRET-restraints. A cluster analysis gives confidence levels for the proposed ensemble of models, and the precision was assessed via bootstrapping. The achieved precisions are significantly better than the uncertainty of the dye position with respect to the macromolecule. The structure of the 4WJ was compared with FRET restrained structures of related RNA 3WJs, where one stem was removed. The types of 3WJs were studied: (I) without bulges, (II) with a small bulge (two unpaired nucleotides) and (III) with larger bulge (5 unpaired nucleotides). In conclusion the overall geometry of the RNA helices depends drastically on the junction type.[1] Sisamakis, E., et al.; Methods in Enzymology 475, 455-514 (2010)[2] Sindbert, S., et al.; J. Am. Chem. Soc. 133, 2463-2480 (2011)[3] Kalinin, S. et al. Nat. Methods in press

RNA three-way junctions are important ribosomal structural motifs. They are also widely used as building blocks and functional components in nanotechnology applications. Forster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling in combination with molecular dynamics simulations was used to determine the structure of RNA three-way junction (3WJ) without bulges, RNA three-way junction with a small (two unpaired nucleotides) bulge and RNA three way junction with bigger (5 unpaired nucleotides) bulge. In total 81 Donor-Acceptor pairs were measured using single-molecule multi-parameter fluorescence detection (smMFD). This allows us to observe structural changes of the molecule induced by addition of bulge to the initial structure. Rigid body models for the major conformers were obtained by docking rigid double-stranded A-RNA helices explicitly taking into account dye position distributions. This is done many times (1000 iterations) with random starting conformations, yielding all local minima. Obtained models were then refined by all-atom MD simulations [1,2]. First results indicate the presence of two coaxially stacked helices for 3WJs with additional bulges at the junction, and absence of such stacking in case with no bulge. noteworthy the stacked helices are different for the 3WJs with two and five nucleotides in the bulge.Our studies showed that high precision FRET measurements are a valuable tool to complement the structural information obtained by X-ray crystallography or NMR spectroscopy as these techniques are limited in detecting minority conformers.References1. Sindbert S, et al. (2011) Accur ate distance determination of nucleic acids via Forster resonance energy transfer: implications of dye linker length and rigidity. J Am Chem Soc 133(8):2463-2480.2. Kalinin S,et al. (2012) A toolkit and benchmark study for FRET-restrained high-precision structural modeling. Nat. Methods in revision.